Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans

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Abstract
Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of P. denitrificans were purified to the homogeneity from cells grown with glutaric acid as the C source. Glutaryl-CoA dehydrogenase had a MW of 180,000 and was made up of 4 identical subunits with MW of .apprx. 43,000 each of which contained 1 FAD molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0-8.5, a catalytic center activity of .apprx. 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of .apprx. 1.2 and 2.5 .mu.M, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of 2 subunits, only 1 of which contained a FAD molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. dentrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had EPR signals at 2.081, 1.938 and 1.879 G, similar to those of the mitochondrial enzyme.